Project Summary/Abstract The goal of mitosis is to accurately transfer replicated chromosomes from parent cell to each daughter cell. The cell accomplishes this using the mitotic spindle, a dense network of ~105 microtubules and ~200 microtubule associated proteins. Microtubules are dynamic polymers that can switch between states of growing and shrinking. Some of spindle's proteins are molecular motors, which consume energy to move, assemble, or disassemble microtubules. Together, microtubule dynamics and motor activity generate mechanical forces, by which the spindle first aligns and then segregates chromosomes during the course of mitosis. This process facilitates the transfer of genetic information from old to new cells, which is an essential feature of life. That being said, aberrant chromosome movements can lead to daughter cells that have the wrong numbers of chromosomes, an unhealthy condition known as aneuploidy that arises in nearly every cancer type. As such, understanding how the spindle moves chromosomes has medical relevance. However, the density of the spindle and the presence of the cell cortex make it difficult to measure forces on chromosomes and resolve single microtubules in vivo. Therefore, many crucial molecular and mechanical details about how the spindle produces forces to move chromosomes remain unknown, and without this knowledge, our understanding of how cells become aneuploid will be lacking. To overcome the obstacles in vivo, the proposed research uses a novel, cell-free assay to reconstitute in vitro the movements of single chromosomes. Experiments will combine single molecule fluorescence imaging, force microscopy, and biochemical methods to study the molecular mechanisms and mechanics of chromosome movement during early mitosis. First, the mechanisms that drive chromosome movement will be investigated by inhibiting or depleting molecular players and measuring microtubule dynamics. Then, force microscopy will be used to measure forces on chromosomes before and after molecular players are inhibited or depleted, to study how different motor proteins contribute to the forces that move chromosomes. Completion of these experiments will improve our understanding of how the spindle applies forces to move chromosomes during mitosis. In the long term, this knowledge will help us improve our understanding of how cells develop aneuploidy.